Class II (Ia) gene products play critical roles in a variety of T lymphocyte responses. They are the primary stimulating antigens in allogeneic and syngeneic mixed lymphocyte responses, they "restrict" recognition of foreign antigens by L3T4+ lymphocytes, and they control the ability of animals to respond to T dependent antigens (immune response [Ir] gene function). A combination of immunological and molecular genetic approaches is being used to gain an understanding of the structural basis for this recognition of Ia by T lymphocytes. Towards this goal, genomic or cDNA clones of various allelic forms of Abeta, Ebeta, Abeta, and Alpha have been isolated and, where necessary, sequenced. Aalpha and Abeta genes have been transfected into B lymphomas or L-cells, and Ia expressing transformants obtained. These have been used to stimulate a variety of T cell hybridomas and clones, establishing the importance of both Aalpha and Abeta polymorphic regions in forming restriction elements. Exon-shuffling between allelic Abeta genes localized both serologic and T cell recognition sites to the highly variable Beta1 domain. Sequence analysis of EMS induced Abeta mutants also localized a critical site of function to a small region of the Beta1 domain near the recently sequenced bm12 mutation. Finally, attempts to construct transfectants expressing "hybrid" I-A molecules revealed an unexpected restriction on alpha:beta chain assembly which maps to the Beta1 domain. These results have shown the validity of this approach in determining the critical structural features of class II molecules recognized by T lymphocytes, and provided new insights into the molecular basis of Ia chain assembly.